Halogenated surface with reduced protein interaction

ABSTRACT

A method for treating a solid surface exposable to protein solutes is provided that reduces interactions of the protein solutes with the surface. Thus, a small bore capillary tube, useful for electrophoretic separation, comprises a reduced interaction phase coated along the bore that includes a terminal moiety covalently bound in the reduced interaction phase through at least one heteroatom. This terminal moiety includes a plurality of halogen atoms, and preferably is an aryl pentafluoro.

FIELD OF THE INVENTION

The present invention generally relates to solid surfaces exposed toprotein solutes, and particularly to capillaries used in electrophoreticseparations by capillary zone electrophoresis.

BACKGROUND OF THE INVENTION

Capillary zone electrophoresis ("CZE") in small capillaries (less thanor equal to 75μ) was first demonstrated by Jorgenson and Lukacs, and hasproven useful as an efficient method for the separation of smallsolutes. J. Chromatog., 218 (1981), page 209; Anal. Chem., 53 (1981),page 1298. The separation process relies upon an electroosmosis effectgenerally described as the flow of a liquid in contact with a solidsurface under the influence of a tangentially applied electric field.Attractive factors for electrophoretic separations by capillary zoneelectrophoresis are the small sample sizes, little or no samplepretreatment, and the potential for quantification and recovery ofbiologically active samples.

For example, U.S. Pat. No. 4,675,300, inventors Zare et al., issued June23, 1987 describes theories and equipment for electrokinetic separationprocesses employing a laser-excited fluorescence detector. The systemdescribed by Zare et al. includes a fused silica capillary with a 75μinside diameter.

Unfortunately, one of the single greatest disadvantages of capillaryzone electrophoresis lies when attempts are made to separatemacromolecules such as proteins. Separations of macromolecules by CZEleads to untoward interactions of the biopolymers with the silicacapillary wall.

Jorgensen et al. had noted that separation of model proteins, such ascytochrome, lysozyme and ribonuclease A, in untreated fused silicacapillaries with a phosphate buffer at pH 7 was accompanied by strongtailing, and suggested this might be caused by Coulombic interactions ofthe positively charged proteins and the negatively charged capillarywall. Jorgensen et al., Science, 222 (1983) page 266.

Lauer et al., Analytical Chemistry, 58 (1986), page 166, has reportedthat the Coulombic repulsion between proteins and the capillary wall ofsilica capillaries can overcome adsorption tendencies of the proteinswith the capillary wall. They demonstrated separations of model proteins(ranging in molecular weight from 13,000 to 77,000) by varying thesolution pH relative to the isoelectric point (pI) of the proteins tochange their net charge. However, disadvantages of this approach arethat silica begins to dissolve above pH 7, which shortens column lifeand degrades performance, only proteins with pI's less than the bufferpH can be analyzed, which drastically reduces the range of usefulanalysis, and interactions which are not Coulombic may still occur evenwith proteins bearing a net negative charge due to the complexity ofprotein composition and structure.

Another approach to the problem of biopolymer, or protein, interactionshas been to increase ionic strength. It has been demonstrated that thisconcept works in principle, but heating is also increased as ionicstrength is increased. This heating tends to degrade the efficiency ofseparation.

Yet another approach to the problem of undesirable protein interactionswith the capillary wall has been to coat the eleotrophoresis tube with amono-molecular layer of non-crosslinked polymer. Thus, U.S. Pat. No.4,680,201, inventor Hjerten, issued July 14, 1987 describes a method forpreparing a thin-wall, narrow-bore capillary tube for electrophoreticseparations by use of a bifunctional compound in which one group reactsspecifically with the glass wall and the other with a monomer takingpart in a polymerization process. This procedure results in a polymercoating, such as polyacrylamide coating, and is suggested for use incoating other polymers, such as poly(vinyl alcohol) andpoly(vinylpyrrolidone). However, this method and capillary tubetreatment tends to destroy the electroosmotic flow, and efficiencies arestill rather low. These rather low efficiencies suggest that undesirableprotein-wall interactions are still occurring.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide small bore capillarytubes that are useful for electrophoretic separations of solutesincluding macromolecules, since interactions between the solutes and thebore are reduced considerably and with high efficiencies.

It is another object of the present invention to provide a method fortreating a solid surface intended for exposure to protein solutes inorder to reduce interactions of the solutes with the surface.

Further objects and advantages of the invention will become apparent tothose skilled in the art upon examination of the specification andappended claims, as well as in practice of the present invention.

In one aspect of the present invention, a small bore capillary tube,useful for electrophoretic separations of protein solutes, comprises areduced interaction phase coated along the bore that includes a terminalmoiety covalently bound in the reduced interaction phase through atleast one heteroatom of an intermediate linkage. This terminal moietyincludes a plurality of halogen atoms.

In another aspect of the present invention, a method for treating asolid surface exposable to protein solutes to reduce interactionstherewith comprises modifying the surface by bonding at least onemolecular layer to the surface and contacting the molecular layer withan aryl halogen compound to form an outer molecular layer with halogenmoieties covalently bound therein.

Capillary tubes as described by the present invention have been preparedand used in highly efficient separations for various protein mixtureswith good reproducibility and consistent performance upon repeated use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 3 illustrate electropherograms of a protein mixture on twocolumns of the invention and FIG. 2 of another protein mixture on athird inventive column; and

FIG. 4 illustrates an electropherogram of the same protein mixture as inFIGS. 1 and 3, but using an unmodified (prior art) capillary tube.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a solid surface that is modified so as tohave reduced interactions with protein solutes. One particularlypreferred application is for small bore capillary tubes, such as thetubes used in capillary zone electrophoresis. These tubes are usuallyless than 500μ, more typically about 20μ to about 200μ, in internaldiameter. Other applications include medical uses, such as heart-lungmachines and implants, where surfaces are exposed to protein solutes.For convenience, reference will hereinafter be to a small bore (lessthan about 500 microns) capillary tube with the bore having beenmodified in accordance with the invention.

The modification is whereby a reduced interaction phase is coated alongthe bore, or inside wall, as an interfacial layer between the insidewall of the capillary and the protein solutions when in use. The reducedinteraction phase of such coating is effective to reduce interactionsbetween protein solutes and the bore, preferably while permittingreasonably high electroosmotic flow and resultingq in excellentefficiencies. When this interfacial layer is about four to about sixmolecular layers thick, then it has been found that electroosmotic flowis reasonably high in use for capillary zone electrophoresis; however,fewer molecular layers (so long as at least one) or greater molecularlayers are possible, and may be desirable for particular applications.When bulk molecular layers are coated on the surface, the electroosmoticflow tends to substantially decrease, which is normally not desired in asystem with a single detector with species which migrate towards twoelectrodes.

The reduced interaction phase includes a terminal moiety that iscovalently bound through at least one heteroatom. The terminal moiety isdistal to the surface and covalently bound to the surface by anintermediate linkage including the heteroatom(s). The terminal moietymust include a plurality of halogen atoms, which may be substituents onan aryl group, an alkylaryl group or an alkyl group. Preferably, theterminal moiety is an aryl pentahalo.

Illustrative terminal moieties with halogen atoms substituted on analkyl group are CX₃ --(CX₂)_(n) -- where n is 0 to about 5 and X isselected from hydrogen and halogen with at least two being halogen.

Illustrative terminal moieties with halogen atoms substituted on analkyl aryl are ##STR1## where n is 1 to about 5 and X is selected fromhydrogen and halogen with at least two being halogen.

Illustrative terminal moieties substituted on an aryl group are ##STR2##where X is selected from hydrogen and halogen with at least two beinghalogen.

These terminal moieties are covalently bound to the surface through atleast one heteroatom of an intermediate linkage. The heteroatom isnitrogen, preferably of an amino group, oxygen, preferably of a carbonylgroup, and may also include sulfur. As will be hereinafter more fullydescribed, several heteroatoms may be present and several are preferred.The heteroatoms increase the hydrophilic character of the reducedinteraction phase and, together with the halogen atoms of the terminalmoieties, reduce protein interactions with the capillary tube such ascaused by van der Waals' forces, hydrogen bonding and point charges. Aswill be hereinafter illustrated by formulas illustrating simplifiedmodels of the inventive reduced interaction phase, the surface includessilanol sites at which the coatings are bonded. These silanol sitesinclude an oxygen molecule between the bulk surface and silicon atoms.The heteroatoms of the invention are in addition to such oxygenmolecules. Thus, the intermediate linkages of which the necessaryheteroatoms are a part may be viewed as being between the silicon atomsand the terminal moieties.

Particularly preferred linkages including the heteroatoms and beingintermediate the surface and the terminal moieties are amides, esters,secondary amines, carbamates, carbonates and dithiols includingactivated carbonyls. Some illustrative intermediate linkages are:##STR3##

The reduced interaction phase can be obtained with or without anintermediary leash step. Surface modifications without an intermediaryleash step will now be described.

SURFACE MODIFICATIONS WITHOUT LEASH

When the surface to be modified is silica based, it is first hydratedand then treated with an organo- or chloro-silane having two functionalend groups. The one functional group reacts specifically with the glasswall (when the surface is of fused silica or the like). Thus, one or twoalkoxy groups (such as methoxy, acetoxy, methoxyethoxy or chloro) reactwith the silanol groups in the wall to form a stable, covalently bondedlinkage. Concentrations of silylating reagent in aqueous solution fromabout 0.1 wt. % to about 1 wt. % result in about four to six molecularlayers being bonded to the surface. These about four to six layers arepreferred to ensure there are no remaining unreacted silanol groups butstill to permit a substantial electroosmotic flow. The other functionalgroup of the silylating reagent is a nitrogen nucleophile, an oxygennucleophile or a carbon electrophile.

The modified surface with nitrogen nucleophile, oxygen nucleophile orcarbon electrophile is then reacted with the compound having halogensubstituents. This halogen compound includes an electrophilic species,when the surface has nitrogen or oxygen nucleophiles, and includes anucleophilic species, when the surface has carbon electrophiles.Exemplary reagents for the former situation are pentafluorobenzoylchloride, pentafluorobenzaldehyde, and pentafluorobenzoic acid. Anexemplary reagent for the later situation is a 2,3,4,5,6pentafluoroalkylamine having the structure ##STR4## where n is 1 toabout 5. When n=1, then the 2,3,4,5,6 pentafluorobenzylamine may beprepared by reduction of the nitrile. However, these electrophilic ornucleophilic halogen compounds may be selected from a wide variety ofdifferent compounds. As some further examples are:

2,3,4,5,6-pentafluorobenzhydrol,

Pentaflorobenzonitrile,

2,3,4,5,6-pentafluorobenzyl alcohol,

2,3,4,5,6-pentafluorocinnamic acid,

2,3,4,5,6-pentafluorophenoxyacetic acid,

2,3,4,5,6-pentafluorophenylacetic acid,

DL-1-(pentafluorophenyl)ethanol,

Pentafluorophenylhydrazine,

2,2,3,3,3-pentafluoro-1-propanol,

Pentafluoropropionic acid,

Pentafluoropropionic anhydride,

Pentafluoropyridine,

2,3,4,5,6-pentafluorostyrene,

Pentafluorothiophenol, and

α-Bromo-2,3,4,5,6-pentafluorotoluene.

Exemplary silylating reagents are 3-aminopropyl trimethoxysilane and3-aminopropyl triethoxysilane. The 3-amino group is reactive withactivated carboxylcarbonyls, such as acid halide, anhydride,carbodiimide activated carbonyl or activated ester (such as N-hydroxysuccinimide ester), intrinsically reactive carbonyl (e.g., aldehyde),haloalkyl carbon electrophile (such as α-halocarbonyls andhaloepoxypropanes), or bisoxiranes. For example, the 3-amino group ofthese exemplary reagents may be reacted with pentafluorobenzoyl chlorideor pentafluorobenzaldehyde to form a reduced interaction phase inaccordance with the invention having the respective structuresillustrated by Formulas I and II (shown as simplified models at a singlesilanol site where R indicates continuation of the silane polymer).##STR5##

Other suitable silylating reagents for surfaces desired to have nitrogennucleophiles include:

4-aminobutyldimethylmethoxysilane,

4-aminobutyltriethoxysilane,

(aminoethylaminomethyl)phenethyltrimethosysilane,

n-(2-aminoethyl)-3-aminopropylmethyldimethoxysilane,

n-(2-aminoethyl-3-aminopropyl)trimethoxysilane,

n-2-aminoethyl-3-aminopropyltris(2-ethylhexoxy)silane,

6-(aminohexylaminopropyl)trimethoxysilane,

aminomethyltrimethylsilane,

p-aminophenyltrimethoxysilane,

aminophenyltrimethoxysilane,

3-(1-aminopropoxy)-3,3-dimethyl-1-propenyltrimethoxysilane,

3-aminopropyltris(methoxyethoxyethoxy)silane,

3-aminopropyldimethylethoxysilane,

3-aminopropylmethyldiethoxysilane,

3-aminopropyltris(trimethylsiloxy)silane, and

ω-aminoundecyltrimethoxysilane.

Silylating reagents yielding oxygen nucleophiles include:

3-glycidoxypropyldimehtylethoxysilane,

(3-glycidoxypropyl)methyldiethoxysilane,

3-glycidoxypropylmethyl-di-isopropenoxysilane, and

(3-glycidoxypropyl)trimethoxysilane.

Reaction of oxygen nucleophiles with an activated carboxyl carbonyl,such as an acid chloride or anhydride, results in a reduced interactionphase illustrated by Formula III (again, the reaction being shown as amodel reaction at a single silanol site and is following reaction of theoxygen nucleophile in diol phase with a reagent such aspentafluorobenzoyl chloride or pentafluorobenzoic anhydride). ##STR6##

Where the surface is modified to have a carbon electrophile, then thehalogen compound will include a nucleophilic species, typically anitrogen nucleophile. For example, the surface may be modified bycontacting with (3-glycidoxypropyl)trimethoxysilane. The terminaloxirane may be directly reacted with 2,3,4,5,6 pentafluorobenzylamine,or may be hydrolyzed and then converted to an activated electrophiliccarbon site with a reagent, such as carbonyl diimidazole, ordisuccinimidyl reagent such as N,N'-disuccinimidyl-oxalate or carbonate,or the carbon site may be activated with an adjacent good leaving group,such as an organic sulfonyl halide. The activated carbonyl or activatedcarbon site then may be treated with the nucleophilic aryl halogencompound.

The structures illustrating use of (3-glycidoxypropyl)trimethoxysilaneas a carbon electrophile followed by reaction with 2,3,4,5,6pentafluorobenzylamine (using either the oxirane or after conversion toan oxirane, to an activated carbonyl or to a carbon site activated withan adjacent good leaving group) are illustrated by Formulas IV-V.##STR7##

SURFACE MODIFICATION WITH LEASH

Rather than reacting the surface modified nitrogen or oxygen nucleophileor carbon electrophile immediately with the desired halogen compound, anintermediary leash step may be performed to attach a spacer arm, orleash. This leash is thus part of the intermediate linkage. An advantageof such a leash step is that additional heteroatoms can be incorporatedinto the reduced interaction phase.

Appropriate spacer arms for reaction with the nitrogen nucleophile areelectrophiles, such as activated carbonyl (e.g., acid halide, anhydrideor carbodiimide activated carbonyl or activated ester (such as N-hydroxysuccinimide ester), intrinsically reactive carbonyl (e.g., aldehyde),haloalkyl carbon electrophile (e.g., α-haloacetic acid orhaloepoxypropanes) and with bisoxiranes. Exemplary activated carboxylcarbonyls are succinyl chloride, succinic anhydride, 1,6-hexanoic acidand carbodiimide such asEDAC(1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) or dicyclohexylcarbodiimide, and disuccinimidyl tartarate and dithio bis(succinimidylpropionate). Exemplary aldehydes are glutaralehyde and succinicsemialdehyde. Exemplary haloalkyl carbon electrophiles are α-bromoaceticacid and epichlorhydrin. Exemplary bisoxiranes are ethylene glycoldiglycidyl ether and 1,4 butanediol biglycidyl ether.

A particular advantage of a dithiol leash (illustrated by the exemplaryreagent dithiobis (succinimidyl propionate) is that a reducedinteraction phase having such dithiol heteroatoms permits regenerationof the surface by reduction and reformation under mild conditions.Reaction Scheme 1 illustrates the preparation of such a regenerablesurface. ##STR8## where NHS is N-hydroxy succinimide ester and R' ismethyl or ethyl.

Where the surface has been modified with an oxygen nucleophile (asearlier described), then an intermediary leash step may be performedwith spacer arms reacting as carbon electrophiles, such as activatedcarboxyl carbonyls, α-halo alkyl or epoxide carbon electrophiles.Exemplary activated carboxyl carbonyls are succinyl chloride, succinicanhydride, 1,6-hexanoic acid and carbodiimide (such as EDAC ordicyclohexyl carbodiimide), N-hydroxysuccinimide activated carbonyls(e.g., disuccinimidyl tartarate and dithiobis(succinimidyl propionate)).The latter provides a dithiol leash permitting a regenerable surface asearlier described. Exemplary halo alkyls are α-halocarbonyl (e.g.,α-bromoacetic acid) and halo epoxy propanes (e.g, epibromhydrin andepichlorhydrin). Exemplary epoxides are bisoxiranes such as ethyleneglycol diglycidyl ether and 1,4-butanediol diglycidyl ether.

Where the surface has been modified with a carbon electrophile, then anintermediary leash step may be used where spacers acting as nucleophilesare selected Exemplary nucleophilic reagents are diamines, carboxylicacid amines and dithiols.

Coatings of the invention have resulted in protein separations withefficiencies in the range of 300,000 to about 1,000,000 statisticalmoments. These highly efficient separations have been accomplished withvery low protein to wall interactions (k'), usually less than 0.02. Theelectroosmotic flow rates at these very low k' values are believed to beabout optimum for maximum efficiencies. The narrow peak widths of FIGS.1-3 indicate such high efficiencies.

EXPERIMENTAL

The following examples, methods, materials and results are described forpurposes of illustrating the present invention. However, other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

Example 1 illustrates the simultaneous preparation of three capillarytubes in accordance with the invention without a leash step. Example II(and FIGS. 1-3) illustrate the use of these three capillary tubes.Example III illustrates typical efficiencies that are achievable incapillary tubing of the invention. Example IV illustrates preparation ofan inventive capillary tube with a leash step. Example V illustrates theeffect on electroosmotic flow with different amounts of silyation(multilayer and bulk).

EXAMPLE I

Silica capillary tubing (Polymicro Technologies) of 20μ I.D. was cutinto 100 cm lengths. Three such capillary tubes were then simultaneouslyprepared by coating a four to six molecular layered reduced interactionphase terminating in halogen moieties along the bore, or inner wall, asfollows:

The silica capillary was was first hydrated with 0.1N KOH by pumpingthrough the capillaries at a rate of about 1-2 μl/minute. The wash wascontinued for an 8-10 hour period. The capillaries were then washed for2-3 hours with DI water.

The walls were then reacted with 3-aminopropyltrimethoxysilane (3-APTS).A 1% solution of 3-APTS was prepared and adjusted to pH 3.5 with aceticacid. This solution was pumped through the capillaries at a rate of 1-2μl/minute for one hour in one direction, then the ends were reversed andthe reagent was pumped in the opposite direction for another hour. Thecapillaries were then attached to a manifold connected to a helium tank,and were cured overnight with a flowing stream of helium.

The capillaries were flushed with anhydrous methanol and anhydroustoluene sequentially at flow rates of 1-2μl/minute for 1-2 hours.Meanwhile, since the acyl chloride (pentafluorobenzoyl choride) ismoisture sensitive, it was removed via syringe using techniques suitablefor air sensitve compounds.

A 0.2M solution of the acyl chloride in anhydrous toluene was passedthrough the capillaries at flow rates of 1-2 μl/minute for 2-3 hours.

Following reaction, the columns were washed first with toluene, thenmethanol and then with DI water at flow rates of 1-2μliters/min for 1-2hours each wash. The columns were then equilibrated to the startingbuffer and were ready for use.

Formula I illustrates the composition of the reduced interaction phaseformed by the just described procedure.

EXAMPLE II

Protein mixtures were made in the range of 2 mg/ml solutions in therunning buffer. The running buffer was made from high purity salts, witha 200 mM phosphate buffer and 100 mM potassium chloride. The proteinsused were purchased as lyophilized powders (Sigma Chemical Company).Dimethyl sulfoxide (DMSO) was used as a neutral marker.

The apparatus used was a Hippotronics power supply and an Isco detector.Detection was accomplished by using an HP 3455 A digital volt meter inconjunction with an HP Yectra computer.

All injections were made hydrodynamically by creating a low pressure atthe elution end of the column. Pressure difference required and durationof injection were determined using Poiseulle's equation. Typicalinjection time for a 20μcapillary with a pressure difference of 20 cm ofmercury was 4-5 seconds, resulting in sample volumes of 0.4 to 0.5 nl.All runs were done at 250 v/cm.

FIGS. 1-3 show electropherograms for the three inventive columnsprepared as described by Example I. The unnumbered peak in FIG. 1 andFIG. 3 is the DMSO marker. There was no marker injected in the FIG. 2separation. The FIG. 3 data was taken from a run in which the injectionpoint was 20 cm. farther from the detection point than for FIGS. 1 and2. In none of the FIGS. 1-4 separations was temperature controlled,which is believed to have caused the time variations. However, theprotein separations of FIGS. 1-3 can be seen to be quite consistent, andwith high efficiencies. FIG. 4 shows an electropherogram for a bare(untreated) silica column of the same size as the three inventivecolumns and run using the same conditions. A comparison of the FIG. 4electropherogram with the FIGS. 1-3 electropherograms demonstrates theexcellent resolution of the seven protein constituents by capillarytubes of the invention in contrast to the untreated capillary tube. Theprotein solutes resolved in FIGS. 1 and 3 were as follows (wherenumbered peaks correspond with the numbered proteins):

1. Lysozyme

2. Ribonuclease

3. Trypsinogen

4. Whale Myoglobin

5. Horse Myoglobin

6. Human Carbonic Anhydrase-B

7. Bovine Carbonic Anhydrase-B

The protein mixture used for the FIG. 2 electropherogram included theabove proteins except for human carbonic anhydrase-B.

EXAMPLE III

Typical efficiencies for the protein solutes electrophoreticallyseparated as described by Example I on the inventive capillaries areillustrated by the data of Table 1. These efficiencies are calculated bytheoretical plates according to the technique described by Kucera etal., J. Chrom., 19 (1965) p. 237.

                  TABLE I                                                         ______________________________________                                                              ELUTION                                                                       TIME      EFFICIENCY                                    PROTEINS      pI      (MIN.)    (BY MOMENTS)                                  ______________________________________                                        1.   Lysozyme     11.0    26.8    325,494                                     2.   Ribonuclease 9.6     34.3    501,583                                     3.   Trypsinogen  9.3     37.4    466,374                                     4.   Whale Myoglobin                                                                            8.0     38.0    499,422                                     5.   Horse Myoglobin                                                                            7.4     40.6    512,825                                     6.   Human Carbonic                                                                             6.6     43.7    379,485                                          Anhydrase-B                                                              7.   Bovine Carbonic                                                                            5.9     48.5    482,493                                          Anhydrase-B                                                              ______________________________________                                    

EXAMPLE IV

0.2% glycidoxypropyltrimethoxysilane in 50 ml water was brought to a pHof 3.5 with acetic acid and allowed to polymerize for 10-15 minutes. Six105 cm lengths of silica capillary (30 microns I.D.) were treated bypassing 0.1N KOH (500 microliters) through, then washed (500microliters) with deionized water. The silylating reagent was allowed topass 1/2 hour through each end at 1-2 column volumes per minute. Thecoating was then cured by passing dry He through the tubes overnight.

110 microliters of tresyl in 5.0 mL of anhydrous toluene (0.2M) weretransferred using syringe technique and then 250 microliters was passedthrough the columns. The columns had been dried first with methanol andthen with anhydrous toluene (each pumped through the capillaries at 250microliters) before treating with the tresyl reagent. The tresyl reagentwas flowed at 1-2 column volumes per minute. The capillary ends werethen sealed and the capillaries allowed to stand at room temperature for1 hour. Toluene was then passed through the columns to wash away excesstresyl, again with a flow of 1-2 column volumes per minute.

88 mg of putrescine (1,4 diaminobutane) were dissolved in 5.0 mL toluene(0.2M). 250 microliters of reagent were pumped through the tresylactivated columns. The columns were allowed to sit for one hour. Toluene(250 microliters) was passed through the columns to wash away the excessreagent.

A 0.2M solution of the acyl chloride (pentafluorobenzoyl chloride) inanhydrous toluene was pumped through the amino-glyco phase describedabove (250 microliters). The columns were washed with toluene, methanol,and then water before being equilibrated to the running buffer.

Formula VI illustrates a composition of the reduced interaction phaseformed by the just described procedure (assuming complete conversion ofboth activated sites). ##STR9##

EXAMPLE V

Two columns were prepared with different quantities of 3-APTS. Onecolumn was reacted with a one percent solution of 3-APTS while the othercolumn was reacted with an eight percent solution of 3-APTS. Neithercolumn was further reacted with the halogen compound, since only DMSO asmarker was run to illustrate the effect on electroosmotic flow between amulti-layer and a bulk coating. The running buffer was 0.2M phosphate,pH 7; 150 volts/cm; detection was by UV (219 nm wavelength); injectionwas 5 cm Hg/3 sec. 0.5% DMSO in the running buffer was detected at 21.97minutes with an electroosmotic flow of 0.460 (mm/sec) for the onepercent column and at 133.70 minutes for an electroosmotic flow of 0.099(mm/sec) for the eight percent column. It is believed that optimum flowrates for maximum efficiency are between about 0.5 to about 0.8 mm/sec,although the bulk coated column may be useful for other applications.

Although the present invention has been described with reference tospecific examples, it should be understood that various modification andvariations can be easily made by those skilled in the art withoutdeparting from the spirit of the invention. Accordingly, the foregoingdisclosure should be interpreted as illustrative only and not to beinterpreted in a limiting sense. The present invention is limited onlyby the scope of the following claims.

It is claimed:
 1. A device for electrophoretic separations of proteinsolutes, comprising:a small bore capillary tube having a reducedinteraction phase coated along the bore and effective to reduceinteraction between protein solute and the bore, the reduced interactionphase including a terminal moiety covalently bound in the reducedinteraction phase through at least one heteroatom, the at least oneheteroatom forming a linkage intermediate the bore and the terminalmoiety, the terminal moiety including a plurality of halogen atoms. 2.The capillary tube as in claim 1 wherein the at least one heteroatom isone or more of nitrogen, oxygen or sulfur and the at least oneheteroatom increases hydrophilicity of the reduced interaction phase. 3.The capillary tube as in claim 1 wherein the terminal moiety is aplurality of halogen atoms that are substituents on an aryl group, analkylaryl group or an alkyl group.
 4. The capillary tube as in claim 1wherein the terminal moiety is an aryl pentahalo group.